The primary objectives of the proposed project are (i) determination of the in vivo function of the Drosophila POU factor, Cf1a, during CNS development, (ii) identification of downstream target genes and upstream regulators of the Cf1a gene and (iii) identification and characterization of genes encoding proteins which may interact directly with the Cf1a protein as heterodimers or coactivators. The proposed specific aims are to generate a collection of Cf1a mutant alleles by ems mutagenesis. Hypomorphic, temperature-sensitive or tissue-specific mutations collected in such screens will help to characterize the Cf1a mutant phenotype in detail and deduce specific cellular requirements for Cf1a protein during embryonic development. Mutant strains will be analyzed by immunological labeling for various cellular markers. Hypomorphic or temperature- sensitive Cf1a alleles resulting from mutagenic screens will also be mutagenized to generate enhancer or suppressor mutations in genes encoding proteins which may interact directly with the Cf1a protein. Protein- protein interactions involving the Cf1a protein will also be investigated using a direct binding screen of a lambda-gt11 library using 32P-labeled Cf1a protein fragments as probe to isolate potential coactivators or detect heterodimer formation. In collaboration with three other Drosophila laboratories at Iowa, an enhancer-trap insertion library will be generated and screened for potential Cf1a target genes. Putative candidates will be tested for Cf1a-dependent expression using Cf1a mutant alleles and the ubiquitous expression of the Cf1a protein from an Hsp70-Cf1a insertion strain. Upstream regulators of the Cf1a gene will be identified by a detailed characterization of Cf1a regulatory sequences using previously cloned genomic sequences. DNase footprinting using embryonic nuclear extracts, transgenic expression of modified Cf1a-lacZ fusion genes and a comparison of evolutionarily conserved sequence elements will be combined to identify sequence elements and corresponding DNA-binding factors which are essential for the correct temporal and cell-specific expression of the Cf1a gene. Known genes which may directly regulate Cf1a expression will be examined by analyzing Cf1a protein expression in various mutant backgrounds affecting midline and tracheal development using a previously characterized Cf1a antiserum. POU-domain transcription factors have been shown in other species to be important in the designation of cell lineage identities. By examining POU-factor function in a system highly amenable to genetic and molecular manipulation, such as Drosophila, information gathered concerning the interactions of transcription factors during development can be extrapolated to higher order systems which cannot be as easily examined. A potential relationship between the Cf1a gene and the differentiation of glial and neuronal cells in the developing CNS should be of interest for clinicians investigating various human neurological syndromes which involve degeneration of neurons or myelinating oligodendrocytes.